Antibodies are cornerstone proteins that play a critical role in the first line of defense against environmental intruders including microbes. They are research and diagnosis tools and most importantly can be used as therapeutic compounds. Numerous companies and research centers are today at different stages of development of antibody-based therapy candidates and several antibody drugs are already on the market.
The antibodies may be polyclonal or monoclonal. Methods of producing polyclonal antibodies from various species, including mice, rodents, primates, horses, pigs, rabbits, poultry, etc. may be found, for instance, in (1). Briefly, the antigen is injected in the presence of an adjuvant (for instance, complete or incomplete adjuvant e.g., Freund's adjuvant) and administered to an animal, typically by sub-cutaneous, intra-peritoneal, intra-venous or intramuscular injection. Repeated injections may be performed. Blood samples are collected and immunoglobulins or serum are separated.
The first method of producing monoclonal antibodies was established by Kohler and Milstein (2). This method is also described in detail, for instance, in (3). Briefly, this method comprises immunizing an animal with the antigen in the presence of an adjuvant, followed by the recovery of spleen or lymph node cells, which are then fused with inmortalized cells such as myeloma cells. The resulting hybridomas produce the monoclonal antibodies and can be selected by limit dilutions to isolate individual clones.
The preparation of antibodies has become essential for the evaluation of novel protein functions and in many cases, antibodies have grown to be therapeutic drugs. The interest for antibodies as therapeutic compounds was recently revived by the development of technologies for the generation of human and humanized antibodies. The many genome-sequencing programs in progress are providing a wealth of information necessitating the systematic preparation of antibodies against novel putative protein drug targets. This has created a costly bottleneck in the process of new drug target identification and has emphasized the need for a novel approach to streamline the process of monoclonal antibody preparation.
Indeed, the basic method of preparing monoclonal antibodies has several limitations. For instance, the preparation of hybridoma involves tedious, lengthy and inefficient processes of hybrid cells generation, drug-selection, screening and clonal expansion during which rare antibody-producing cells with the desired antigen specificity may be lost. Another difficulty is that rapid screening methods have to be developed to identify unique antibody-producing hybridoma in a large pool of hybridomas. These methods may vary with the nature of the target antigen and its availability. Yet another difficulty is that hybridomas can mainly be generated using cells from mouse or few other non-primate animals. Therefore, unless mice that are transgenic for the expression of human antibodies are used, hybridomas yield non-human antibodies that need to be transformed into humanized antibodies using recombinant DNA methods. The later process is required for therapeutic applications and can only be initiated once clones producing antibodies with desired specificity are obtained.
To address these limitations, two types of strategies have emerged, consisting of 1) improving specific steps of the classical approach described above and 2) developing antibody repertoires using recombinant DNA technology. The first strategy yielded, for instance, the SLAM technology (4) in which the need for preparation and screening of hybridoma is eliminated. The second strategy yielded, for instance, phage display technologies in which target proteins react with libraries of bacteriophage expressing antibodies or fragment thereof using a bacteriophage panning methods (5). More recently, the HuMY technology was developed by GeneTastix (U.S. Pat. No. 6,406,863) in which fragments of target proteins react with a library of antibody repertoire using a two-hybrid method in yeast.
Although improvements have been made, each method developed so far still has limitations. Notably, a major limitation for most methods is that they require large amounts of target protein for animal immunization and/or screening of hybridoma or bacteriophage libraries. Even in the case of the SLAM technology, which does not require the preparation and screening of hybridoma, large amounts of purified target proteins are still needed to perform plaque assays for isolating antibody-forming cells as described by the authors. The need for purification of recombinant proteins in large amounts hampers the rapid validation of multiple potential drug target proteins. In many instances, purification process varies from protein to protein, yields insufficient amounts of material, non-functional or denatured products. The purification process becomes even more problematic when dealing with membrane proteins or entities consisting of multiple polypeptide complexes.
Some phage display as well as two-hybrid methods use libraries of antigenic peptides and, thereby, alleviate the need for large-scale antigen purification. However, one deficiency with these methods is that antibodies against conformational epitopes are difficult to obtain and for instance, antibodies restricted to epitopes rising from multi-polypeptide entities such as anti-MHC/peptide complex antibodies cannot be obtained. Another limitation is that the sizes and qualities of the peptide libraries and of the antibody libraries used do not always allow isolating antibodies with high antigen-specific affinity. This is because affinity maturation of antibodies can only be performed if a first generation antibody is obtained. In contrast, the unlimited repertoire of antibody sequences found in mammals combined with the natural affinity maturation process occurring following repeated immunization of these animals provides the most efficient way to potentially generate antibodies against any epitope.
The present invention addresses the limitations of antibody preparation methods by providing a novel and effective approach. It also provides new valuable tools to improve existing methods of antibody preparation.